Conventionally, optical microscope techniques have been widely used for a long period of time, and various types of microscopes have been developed. In recent years, with the advancement of peripheral techniques thereof such as a laser technique and an electronic graphic technique, microscope systems having further high performance have been developed.
In view of the background as described above, there is proposed a high-performance microscope that can not only control a contrast of an image to be obtained, but also perform a chemical analysis by using a double resonance absorption process induced by illuminating a specimen with lights of plural wavelengths (for example, see Patent Literature 1).
This microscope selects, by using the double resonance absorption, a specific molecule, and observes absorption and fluorescence caused by a specific optical transition. This theory will be described with reference to FIGS. 14 through 17. FIG. 14 shows electronic structures of valence orbits of molecules constituting the specimen. First, an electron in a valence orbit owned by the molecule in a ground state (S0 state: stable state) illustrated in FIG. 14 is excited to a first excited state (S1 state) illustrated in FIG. 15 by a light of a wavelength λ1. Then, similarly, excitement is performed to a second excited state (S2 state) illustrated in FIG. 16 by a light of another wavelength λ2. With this excited state, the molecule returns to the ground state as illustrated in FIG. 17 while emitting fluorescence or phosphorescence.
In a microscope method using the double resonance absorption process, an absorption image or luminous image is observed by using the absorption process in FIG. 16 or the emission of the fluorescence or phosphorescence in FIG. 17. With this microscope method, a molecule constituting a specimen is first excited to the S1 state as illustrated in FIG. 15 with the light of resonance wavelength λ1 by a laser beam and the like, and at this time, the number of molecules in the S1 state in a unit volume increases as the intensity of the irradiation light increases.
A linear absorption coefficient is given as a product of an absorption cross-section per molecule and the number of molecules per unit volume, and hence, in the exciting process as illustrated in FIG. 16, the linear absorption coefficient for the subsequently irradiated resonance wavelength λ2 strongly depends on the intensity of the first applied light of the wavelength λ1. That is, the linear absorption coefficient for the wavelength λ2 can be controlled with the intensity of the light of the wavelength λ1. This indicates that a contrast of the transmission image can be completely controlled with the light of the wavelength λ1 by irradiating the specimen with the lights of the wavelength λ1 and the wavelength λ2 to obtain the transmission image by the wavelength λ2.
When the deexcitation process by fluorescence or phosphorescence from the excited state of FIG. 16 to the ground state shown in FIG. 17 is possible, the luminous intensity is proportional to the number of molecules in the S1 state. This makes it possible to control an image contrast, even when used as a fluorescent microscope.
Further, this microscope method using the double resonance absorption process can be used not only for controlling the image contrast described above but also for performing the chemical analysis. That is, since the outermost valence orbit illustrated in FIG. 14 has an energy level intrinsic to each molecule, the wavelength λ1 is different for molecules. At the same time, the wavelength λ2 is also intrinsic to each molecule.
When the specimen is illuminated with the conventional single wavelength, it is possible to observe the absorption image or fluorescent image of a specific molecule in some degree. However, in general, since some molecules overlap their wavelength ranges of absorption band with each other, it is impossible to accurately identify the chemical composition of the specimen when the specimen is illuminated with the single wavelength.
On the other hand, according to the microscope using the double resonance absorption process, the molecules to absorb or illuminate the light are defined by using the two wavelengths of the wavelength λ1 and the wavelength λ2, so that the chemical composition of the specimen can be identified more accurately than the conventional method. In a case where the valence electron is to be excited, only a light having a specific electric-field vector with respect to a molecular axis is intensively absorbed. Thus, it is possible to identify the direction of orientation even for the same molecule when an absorption image or a fluorescence image is obtained while determining the polarization directions of the wavelength λ1 and the wavelength λ2.
In recent years, there has been proposed a fluorescent microscope which has a high spatial resolution exceeding a diffraction limit by using the double resonance absorption process (for example, see Patent Literature 2).
FIG. 18 is a conceptual diagram illustrating the double resonance absorption process of molecules, in which a molecule in the ground state S0 is excited to the first excited state S1 with the light of the wavelength λ1 and further to the second excited state S2 with the light of the wavelength λ2. Note that FIG. 18 illustrates that fluorescence from this second excited state S2 is extremely weak for some kind of molecules.
The molecule having such optical properties as illustrated in FIG. 18 experiences a remarkably interesting phenomenon. FIG. 19 is a conceptual diagram illustrating the double resonance absorption process similar to FIG. 18, in which the X axis of abscissa represents an extension of a spatial distance, and a spatial area A1 which is irradiated with the light of the wavelength λ2 and a spatial area A0 which is not irradiated with the light of the wavelength λ2 are shown.
In FIG. 19, a great number of the molecules in the S1 state are generated in this spatial area A0 by the excitation with the light of the wavelength λ1, and at this time, a fluorescent light emitted from the spatial area A0 with a wavelength λ3 can be observed. In the spatial area A1, however, the irradiation of the light of the wavelength λ2 excites most of the molecules in the S1 state instantly to the S2 state at a higher level, so that the molecules in the S1 state disappears. The phenomenon as described above is confirmed in several molecules. As a result, the fluorescence of the wavelength completely disappears in the spatial area A1, and further, the fluorescence from the S2 state does not exist intrinsically, so that the fluorescence itself is completely inhibited in the spatial area A1 (fluorescence inhibiting effect). Therefore, the fluorescence exists only in the spatial area A0.
Considered from the application field of the microscope, this result has a remarkably important meaning. That is, in the conventional scanning type laser microscope, a laser beam is condensed to produce a micro beam with a condensing lens, thereby to scan a target specimen. In this case, the size of the micro beam is determined by the diffraction limit which is determined by a numerical aperture of a condensing lens and a wavelength. As a result, a higher spatial resolution cannot be expected in principle.
However, in a case of FIG. 19, the fluorescent area is inhibited by partially overlapping two types of lights, light of the wavelength of λ1 and light of the wavelength of λ2, in a spatial manner, and hence, by paying attention to the irradiation area of the light of the wavelength of for example, the fluorescent area can be made narrower than the diffraction limit which is determined by a numerical aperture of a condensing lens and a wavelength, which makes it possible to substantially improve the spatial resolution. Hereinafter, the light of the wavelength of λ1 is referred to as a pump light, and the light of the wavelength of λ2 is referred to as an erase light. Therefore, by adopting this principle, it is possible to realize a super-resolution microscope such as a super-resolution fluorescent microscope, which exceeds the diffraction limit and utilizes the double resonance absorption process.
For example, in a case where the Rhodamine 6G dye is employed, the molecule of the Rhodamine 6G is excited from the S0 state to the S1 state by irradiating the light (pump light) of the wavelength of 532 nm, thereby emitting the fluorescent light having a peak at the wavelength of 560 nm. At this time, when the light (erase light) of the wavelength of 599 nm is irradiated, the double resonance absorption process occurs, and the molecule of the Rhodamine 6G transit to the S2 state where the fluorescent light is less likely to be emitted. That is, the fluorescent is inhibited by irradiating the pump light and the erase light to the Rhodamine 6G at the same time.
FIG. 20 is a configuration diagram illustrating a main portion of the conventionally proposed super-resolution microscope. This super-resolution microscope is based on an ordinary laser scanning type fluorescent microscope, and is formed mainly by three independent units, namely, a light source unit 210, a scan unit 230, and a microscope unit 250.
The light source unit 210 has a light source 211 for a pump light, and a light source 212 for an erase light. The pump light emitted from the light source 211 for the pump light is inputted into a dichroic prism 213, is reflected by the dichroic prism 213, and then, is emitted. The erase light emitted from the light source 212 for the erase light is polarized by the polarizing element 214; a phase of the thus polarized light is modulated by a phase plate 215; the modulated light is inputted into and passes through the dichroic prism 213; and, the passing light is combined with the pump light on the same axis, thereby being emitted.
In a case of observing the specimen dyed with the Rhodamine 6G dye, the light source 211 for the pump light is configured such that Nd: YAG laser is employed, and a light of the wavelength of 532 nm, which is a second harmonic wave of the light from the laser, is emitted as the pump light. Further, the light source 212 for the erase light is configured such that Nd: YAG laser and a Raman shifter are employed, the second harmonic wave of the Nd: YAG laser is converted into the light of the wavelength of 599 nm by the Raman shifter, and the converted light is emitted as the erase light.
The phase plate 215 is configured such that phase differences of the erase light make 2π-turn around an optical axis. For example, as illustrated in FIG. 21, the phase plate 215 has independent eight areas around the optical axis, and is formed by etching a glass plate such that the respective phases are shifted by ⅛ of the wavelength of the erase light. By condensing the light passing through the phase plate 215, a hollow-shaped erase light in which electric fields are cancelled at the optical axis can be generated.
In the scan unit 230, the pump light and the erase light emitted from the light source unit 210 on the same axis are made pass through a half prism 231, and, the lights are swingingly scanned in a two-dimensional direction by two galvano mirrors 232 and 233, and are outputted to the microscope unit 250, which will be described later. Further, the scan unit 230 is configured such that: the fluorescent light arriving from the microscope unit 250 traces the reverse path to the ongoing path, and is separated by the half prism 231; the separated fluorescent light passes through a projection lens 234, a pinhole 235, and notch filters 236 and 237; and, the light is received by a photomultiplier 238.
In FIG. 20, the galvano mirrors 232, 233 are illustrated so as to be swingable in the same plane for the purpose of simplifying the drawing. Note that the notch filters 236 and 237 are used for removing the pump light and the erase light contained in the fluorescent light. The pinhole 235 is an important optical element serving as a confocal optical system, and allows only the fluorescent light emitted at a specific fault plane existing in a target specimen to pass through.
The microscope unit 250 is a so-called ordinary fluorescent microscope, in which the pump light and the erase light arriving from the scan unit 230 are reflected by a half prism 251, and are condensed by a microscope objective lens 252 on an target specimen 253 containing a molecule in three electronic states including at least the ground state. Further, the fluorescent light emitted from the target specimen 253 is collimated by the microscope objective lens 252 again, is reflected by the half prism 251, and is returned again to the scan unit 230, and part of the fluorescent light passing through the half prism 251 is guided to an eyepiece lens 254 so as to be able to make the visual observation as the fluorescence image.
According to the super-resolution microscope described above, it is possible to inhibit the fluorescent light other than the light in the vicinity of the optical axis where the intensity of the erase light is zero at the light condensing point of the target specimen 253, and, as a result, measure only a fluorescent-labeler molecule existing in a region narrower than the extension of the pump light. Therefore, by arranging fluorescent signals of the respective measurement points in a two-dimensional manner by using a computer, it is possible to form a microscope image having a resolution higher than the spatial resolution of the diffraction limit.
Incidentally, the super-resolution microscope using an optical response with two colors of illumination described above strongly depends on polarization, and an expected optical property cannot be obtained when a predetermined polarization condition is not satisfied. For example, it has been known that the above-described fluorescent inhibiting effect strongly appears when the polarization directions of the two colors of illumination lights are the same, and the effect is weakened when the polarization directions are orthogonal to each other (see, for example, Non-Patent Literature 1).
Further, a polarization state greatly affects a beam shape of the erase light. That is, in a case of employing the phase plate 215 shown in FIG. 21, the beam shape varies depending on the polarization state. For example, it has been known that, in a case where the erase light is converted into a circularly polarized light by the polarizing element 214 and then is condensed with a lens having a high numerical aperture, a polarization component in the optical axis direction occurs in accordance with the turning direction of the electric field in the circularly polarized light, and, the intensity at the center does not become zero, whereby it is impossible to obtain the beam shape having the hollow structure (see, for example, Non-Patent Literature 2). This is because, when a light having the orthogonal electric-field vectors in a plane is condensed, electric-field vector components parallel to the optical axis direction are generated independently of each other; and, the components are added one after another when these components are parallel, although the intensity at the center becomes zero when these are antiparallel.
As described above, when the erase light does not have the hollow-shaped beam on the focal plane, the fluorescence of the pump light is inhibited at the center portion, which leads to deterioration in the spatial resolution. Therefore, it is necessary to optimize the polarization state of the pump light and the erase light in order to obtain a desired super-resolution effect.
Further, in order to obtain the sufficient super-resolution effect, it has been known that it is necessary for the hollow portion of the erase light and the peak point of the pump light to coincide with each other on the focal plane with an accuracy of about 30 nm (see, for example, Non-Patent Literature 3).
Yet further, in a case of employing the phase plate 215 shown in FIG. 21, the electric field on the optical axis cannot be cancelled on the focal plane, and an ideal hollow spot cannot be obtained, when the center portion of the phase plate 215 and the center portion of the erase light are not completely matched, that is, when the phase of the erase light is not completely reversed at a position symmetrical with respect to the optical axis. As a result, the super-resolution effect cannot be obtained.
Therefore, conventionally, the polarizing element 214 and the phase plate 215 are configured so as to be independently adjustable, in order to obtain the super-resolution effect.
Conventional Art References
Patent Literatures
Patent Literature 1: Japanese Patent Application Laid-open No. 8-184552
Patent Literature 2: Japanese Patent Application Laid-open No. 2001-100102
Non-Patent Literatures
Non-Patent Literature 1: N. Bokor, Y. Iketaki, T. Watanabe, K. Daigoku, N. Davidson and M. Fujii, Opt. Comm., 272 (1), (2007) 263-269.
Non-Patent Literature 2: Y. Iketaki, T. Watanabe, N. Bokor and M. Fujii, Opt. Left., 32 (2007) 2357-2359
Non-Patent Literature 3: Y. Iketaki, T. Watanabe, N. Bokor and M. Fujii, e-Journal of Surface Science and Nanotechnology, 6 (2008) 175-179